PD-1 Knockout Aggravates Motor Dysfunction In The MPTP Model of Parkinson's Disease By Inducing Microglial Activation And Neuroinammation In Mice

Background: Abundant microglial reaction and neuroinammation are typical pathogenetic hallmark of brains in Parkinson’s disease (PD) patients, but regulation mechanisms are poorly understood. In this study, the promoting effects of PD-1-diciency on microglial activation, neuroinammation and motor dysfunction were identied using PD animal model. Methods: Using C57 wild-type (WT), PD-1 knockout (KO) and MPTP model, we designed WT-control, KO-control, WT-MPTP and KO-MPTP groups. Motor dysfunction of animal, distribution of PD-1-positive cells, dopaminergic neuronal survival, glial cell activation and generation of inammatory cytokines in midbrains were observed by behavior detection, immunohistochemistry and western blot methods. Results: Microglial cells showing PD-1/Iba1 double-positivity were numerously distributed in the substantia nigra of control whereas they decreased in MPTP model. Compared with WT-MPTP, KO-MPTP mice exacerbated in their motor dysfunction, decreased level of TH expression and decreased TH-positive neuronal protrusions. Microglial cell activation and expression of proinammatory cytokine iNOS, TNF-α, IL-1β and IL-6 signicantly increased, and levels and phosphorylation of AKT and ERK1/2 were also elevated in KO-MPTP mice. Conclusions: PD-1 knockout could aggravate motor dysfunction of MPTP mouse model by promoting microglial activation and neuroinammation in midbrains, suggesting that PD-1 signaling abnormality might be involved in PD pathogenesis or progression. interleukin-6;

natural killer cells, and plays an important roles in regulating antigen response or limiting host adaptive immune response by threshold of T cells and B cells, and thereafter joining in prevention of autoin ammation response or diseases (16-18). PD-1 ligands include PD-L1 and PD-L2 bind PD-1 receptor, initiate PD-1 signaling and followed cellular effects. In the tumor microenvironment and in ammatory state, expression of PD-1 in T-cell increases abnormally. PD-L1 then combines PD-1 and initiates a series of inhibition signaling, resulting in decline of T-cell function, causing immune tolerance of tumor cells and evading T-cell immune surveillance (19,20). Therefore, PD-1/PD-L1 signaling pathway is critically in immune function regulation and immune tolerance, and provides a therapeutic way for immune modulatory therapy or intervention of various diseases (21)(22)(23)(24).
It is well known that microglial cells belong to the monocyte-macrophage system based on their source of occurrence and characteristics, showing antigen presentation function and secretion of a variety of active factors. PD-L1 is identi ed in the glial cells and expressional change is observed under CNS in ammatory state (24,25). However, it is still lack of studies on PD-1/PD-L1 working in the neurodegenerative diseases, especially relation to microglial activation and neuroin ammatory response. It is interesting to note that existing studies on PD-1 role in the experimental autoimmune encephalomyelitis and spinal cord injury, and it reveals that PD-1-de ciency results in an increased neuropathological in ammation (26)(27)(28)(29). Based on literature evidences, we speculate that PD-1 signaling pathway may be also an potential regulating factor in the activated glial cells and in ammation and neuronal damage during pathogenesis of PD. Therefore, PD-1 knockout and MPTP mouse model were applied in this study, immunohistochemistry, western blot and motor behavior detection were used to observe effects of PD-1de ciency on glial cell activation, neuroin ammatory reaction and motor dysfunction of animals, for purpose to determine possible mechanistic involvement of PD-1 signaling pathway in PD pathogenesis or progression, which may thereafter provide a new basis for exploring immunotherapy potentially for PD.

Preparation of MPTP animal model
The C57BL/6 mice, Wild-Type and PD-1 knockout weighing 20 ± 2g, male and female, were used. The animal were arranged in WT control (WT-CON), WT MPTP (WT-MPTP), PD-1 knockout control (KO-CON), and PD-1 knockout MPTP (KO-MPTP) group with 5-10 mice each (n = 5-10). In MPTP group (WT-MPTP and KO-MPTP group), MPTP 30 mg/kg/day was intraperitoneally injected for consecutive 5 days. In control group (WT-CON and KO-CON) same amount of 0.9% saline was given by an intraperitoneal injection. After motor behavior detection of animals, the perfusion-xation and frozen sections of midbrains were performed for immunohistochemical staining or double-labeling, or sample extraction of fresh midbrain tissues were carried out for western blot respectively.

Animal motor behavior detection
The MPTP animal model were prepared at time-points of the 5th, 10th and 15th day, groups of WT-CON, KO-CON, WT-MPTP, KO-MPTP mice were tested for motor behavior. Using the boom experiment method, the movement time (Seconds) and the number of falls (per 5min) were observed and counted, and the movement dysfunction and changes in different experimental groups of mice were analyzed and shown. Immunohistochemistry Animals in injectable anaesthetic state, saline and fallowed 4% formaldehyde solution was perfused for brain xation, the midbrains was put into 20% sucrose PB solution, soaked overnight to tissue subsidence, the midbrain samples containing nigral region were token for continuous frozen sections with 15-20 micron thickness. Sections were selected to be rinsed 3 times in 0.01M PBS buffer and then incubated in 0.1% Triton X-100-3% donkey serum for 30 min. Sections were incubated in primary antibody solution respectively for 24hours at 4℃ and 1h at room temperature. After rinsing 3 times in 0.01M PBS, sections were incubated in secondary uorescence-labeled antibody solution for 4hours at room temperature. Finally Midbrain sections were processed for DAPI nuclear counter-stain and observed under a laser scanning confocal microscope (LSCM, Olympus, FX-1000), and interested images were captured for demonstration representatively.

Western blot
Western blot was performed as in our previous studies (Cheng et al., 2020). Fresh lysates from midbrain samples were made in lysis buffer (Beyotime Biotechnology). The protein concentrations were measured by BCA, and 10µl (2µg/µl) of sample solution was loaded for each panel. After separation in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (10%), protein samples were transferred onto PVDF membranes (Millipore). Membranes were then blocked with 5% nonfat milk/TBST (0.1% Tween-20, TBS) for 1hour and incubated with primary antibody respectively at 4°C overnight. The β-actin level was used as an internal control for immunoblot. After incubation with HRP-conjugated secondary antibody at room temperature for 1 h, immunoblots were detected using an advanced ECL detection kit according to manufacturer's instruction. For re-probing, the transfer membrane was stripped by complete washing with stripping buffer (glycine-SDS buffer, pH 2.0) for 15 min, followed by TBST (pH 7.4) three times, and then processed for a standard probing process. Immunoblot images were acquired and analyzed using Bio-Rad's system. Quanti cation was performed and presented as a density ratio to internal control.

Statistical Analysis
Quantitative data on immuno-positive cell counts and immunoblots were shown by Mean ± Standard Error (mean ± SEM), and the differences between the groups were compared by Student T-test (between two groups), and One-way ANOVA and Dunnett post-test (between four groups) using GraphPad Prism (5.0) software. When the P-value of comparison between groups was less than 0.05 and considered to have statistical signi cance.

Distribution of microglial cells showing PD-1 immunoreactivity in the substantia nigra
First, double immuno uorescence for PD-1/Iba1, PD-1/GFAP and PD-1/NeuN was carried out to observe localization of PD-1-immunoreactivity in the microglial cells, astrocytes and neurons in midbrains under a laser scanning confocal microscope. PD-1/Iba1 double-labeled microglial cells were observed and numerously distributed in the substantia nigra of mice, and PD-1-immunoreactive products were clearly and strongly localized in cell membrane of the microglial cells. On the other hand, PD-1/GFAP doublelabeled astrocytes, PD-1/NeuN double-labeled neuronal cells were not observed in the substantia nigra regions (Fig. 1). In addition, double immuno uorescence for PD-L1/Iba1, PD-L1/GFAP, PD-L1/NeuN was also performed to observe if any PD-L1, PD-1 ligand, was localized in the microglial cells, astrocytes and neurons, and it indicated that PD-L1 was also mainly located in the microglial cells rather than astrocytes and neurons (data not shown here).
Decrease of microglial cells with PD-1/Iba1 double-labeling in the substantia nigra of MPTP model Then, immunohistochemstry was again applied to observe if any changes of PD-1/Iba1 double-labeled microglial cells distributed in the substantia nigra occurred in animal model of Parkinson's disease. A sub-acute mouse model of Parkinson's disease i.e. MPTP 30 mg/kg for continuous 5 days was performed and followed by nigral sections and immunostaining. Both Iba1-immunopositive microglial cells and GFAP-positive astrocytes in MPTP model showed active morphology with an increase in the numbers, consistent with our previous observations. Compared with control group, the number of microglial cells with PD-1/Ibal double-labeling decreased signi cantly in MPTP group, and immunostaining intensity of PD-1 also decreased obviously. Comparison of corresponding ngral staining regions in control group and MPTP group showed differences in their morphology and quantity of PD-1/Ibal double-labeled cells (Fig. 2). Cell count on PD-1/Iba1 double-labeled cells in the unit area of onesided nigral sections indicated that 280 ± 32 in control group and 189 ± 26 in MPTP group with signi cant statistical differences (Student T-test, P < 0.01).
Aggravation effect of PD-1 knockout on motor dysfunction and reduction of TH-positive protrusions in MPTP model After that, PD-1-knockout mice was introduced to assess effect of PD-1 on the motor function and survival of dopamine neurons in MPTP model by comparing animal movement functions (motion time and falling times) and immunohistochemical visualization of TH nigral neurons at time-points of the d5, d10, d15 in WT-CON, WT-MPTP, KO-CON, KO-MPTP group. Compared with WT-MPTP, movement dysfunction in KO-MPTP group was more severe, the performance was more slow, the movement time was obviously shortened, the falling times increased, or animals was unwilling to move or even stay in place. The WT-CON mice also showed a decrease in motion time compared to KO-CON group, but there was no signi cant change in the falling times (Fig. 3). Immunohistochemistry and Western blot were further carried out to show TH-positive neurons, neuronal protrusions and TH expression levels. The number of TH-positive neurons in WT-MPTP group decreased signi cantly compared with WT control, which was same as the previous observations. Compared with WT-MPTP group, there was no signi cant difference in number of TH-positive neurons, but KO-MPTP showed a decreased trend of TH-positive neuronal cell protrusions or processes than WT-MPTP group (P < 0.05) (Fig. 4A, B). Immunoblot con rmed that KO-MPTP mice showed a signi cant decrease in TH expression level compared with WT-MPTP group (P < 0.01) (Fig. 4C).
Promotion effect of PD-1 knock-out on microglial activation and proin ammatory cytokine expression in MPTP model Furthermore, effects of PD-1 knockout state on glial cell activation, pre-in ammatory factor (M1 marker molecule) and anti-in ammatory factor (M2 marker molecule) expression levels were analyzed to evaluate PD-1 participation in neuroin ammatory response. The KO-MPTP mice had a signi cant increase in Iba1 expression compared to WT-MPTP group (P < 0.01), while expression of PD-1, PD-L1 and GFAP did not show signi cant changes (P > 0.05) (Fig. 5). Expressions of proin ammatory cytokine iNOS, TNF-α, IL-1β and IL-6 showed up-regulated trend in both KO-CON and KO-MPTP group. The KO-MPTP mice increased expression of iNOS, TNF-α, IL-1β and IL-6 signi cantly compared to the WT-MPTP group. The KO-CON group also showed upward changes of iNOS, IL-1beta, and IL6 except of TNF-α compared to WT-CON group (Fig. 6). Double immunostaining showed positive localization of iNOS, TNF-α, IL-1β and IL-6 in the microglial cells (data not shown). In addition, anti-in ammatory cytokine ARG-1, TGF-β, IL-4, IL-10 expression change trend was not fully consistent. Compared with WT-MPTP group, ARG-1 showed a signi cant downward change, TGF-β and IL-4 no signi cant change, while IL-10 showed a clear upward trend in expression in KO-MPTP group (Fig. 7). These data indicated that PD-1 knockout increased abnormal glial activation and proin ammatory cytokine production, and might thereafter induce or aggravate neuroin ammatory response, dopaminergic neuronal damage and motor dysfunction in MPTP animal model.

Motivation in uence of PD-1 knockout on AKT and ERK1/2 signaling in MPTP model
Finally, Western blot was applied to analyze possible cell signaling mechanism of PD-1 knockout affecting glial activation and in ammatory response by examining AKT, p-AKT, ERK1/2 and p-ERK1/2 expression in the substantia nigra of MPTP animal model. Compared with WT-MPTP group, p-AKT, tAKT, p-ERK1/2 and tERK1/2 showed an increase in the expression levels with statistical signi cant in KO-MPTP group (Fig. 8). Compared with WT-MPTP, at the same time, the ratio of p-AKT to tAKT, or p-ERK1/2 to tERK1/2 also increased signi cantly in KO-MPTP group. Data showed that phosphorylation or activation level of AKT and ERK1/2 increased to a certain extent in KO-MPTP group, indicating that PD-1 knockout could possibly promote activation of AKT and ERK1/2 signaling pathways in MPTP model of PD. However, whether AKT, p-AKT, ERK1/2, p-ERK1/2 signal molecules were exactly or mainly localized in these activated microglial cells or astrocytes was subject to further experimental con rmation.

Discussion
In this study, PD-1 knockout mice and MPTP model of Parkinson's disease, motor behavior detection, immunohistochemistry and western blot were used to observe and analyze role of PD-1 signaling in brain glial activation, in ammation response and movement dysfunction of Parkinson' animals. It revealed that PD-1/Iba1 double-positive microglial cells numerously distributed in control, whereas they were signi cantly reduced in the MPTP model. Compared with WT-MPTP, KO-MPTP group mice showed signi cant increases in motor dysfunction, decreased expression level of TH protein and decrease in THpositive neuronal protrusions, accompanied by activation of microglial cells and astrocytes, increased expressional levels of proin ammatory cytokine iNOS, TNF-α, IL-1β and IL-6. Further detection showed that PD-1 knockout induced elevated expression and phosphorylation activation of AKT and ERK1/2. The results indicated that PD-1 knockout aggravated animal motor dysfunction of PD model, possibly by promoting microglial cell activation, increasing expression of proin ammatory cytokines and triggering AKT and ERK1/2 signaling in the substantia nigra, suggesting that PD-1 signaling abnormality may be involved in micrioglial activation and neuroin ammation response in Parkinson's disease progression.
Many studies showed that abnormal activation of microglial cells and astrocytes in the CNS dominated neuroin ammatory reaction, which might be a critical factor leading to the development of various neurodegenerative diseases such as AD, MS and PD (4)(5)(6)(7)(8). In the state of in ammatory reaction, microglial cells functioned as CNS inherent immune cells, activated microglial cells initiated differentiation in two opposite directions, namely, in ammatory state (M1), anti-in ammatory state (M2) functional polarization (29). On the one hand, microglial cells in M2 polarization could remove pathogens or cell fragments and protect from damage to the brains. On the other hand, microglial cell with M1 polarization was in an in ammatory state and could produce a series of cytokines, and thereby further activating astrocytes, being collaborative in ammatory response. The astrocytes regulated the immune response and react to pathological changes by hypertrophy, presented as functional activated state, the activated astrocytes also occurred in similar functional differentiation, that is, in ammatory state (A1) and anti-in ammatory state (A2) polarization, activated A2 astrocytes promoted tissue repair and help maintain function of the central neurons. The activated A1 astrocytes secreted a large number reactive oxygen and pro-in ammatory cytokines, affecting neurons and other glial function, triggering a vicious circle, exacerbating the biological process of ampli cation of in ammatory reactions and neuronal damage in the CNS (30,31).
It was known that as an important inhibitory immune checkpoint, PD-1 is the regulating molecule of immune cell function (32). Data showed that PD-1 and PD-L1 expression levels changes with healthy and pathological state in the CNS, for example, 20% of microglial cells expressed PD-L1 in uninfected normal mice, while more than 90% of microglial cells showed induced PD-L1 expression 1week after infection (24,25). Stimulation of interferon-gama (IFN-γ), autoimmune diseases, brain tumors and stroke state induced central T-cell activation and PD-1 production. Increased PD-L1 binding and activation of PD-1 signaling pathway regulated tumor microenvironment and in ammatory response, and thus affected progression of above diseases (33). Some studies indicated that PD1/PD-L1 signaling activation promoted differentiation of microglial cells into anti-in ammatory states (M2) and reduced secondary brain damage in the cerebral hemorrhage (34,35). In addition, many studies focusing on brain tumors showed that tumor cells secreted high level of PD-L1, thereafter induced T cells to produce high level of PD-1 molecule. Combination of PD-1/PD-L1 or PD-1 signaling activation caused decrease or "failure" state of T-cell function, resulting in the migration and diffusion of tumor cells. As a targeted anti-tumor strategy, therefore, PD-1-based inhibitors (PD1 antibody) have attracted more and more attention and successfully used in clinical.
Whether PD-1 signaling pathway was involved in glial cell activation and in ammation of Parkinson's disease, which in turn affected course of the disease, was major concern of this study. Present results showed that the absence state of PD-1 leaded to more obvious glial activation in the MPTP model, increased the expression level of in ammatory factors, and aggravated motor dysfunction, which indicated that PD-1 had a certain restrictive effect on neuroin ammation dominated by activated glial cells, and played a neuroprotective role by regulating or limiting in ammatory response. Our result was supported by studies of Yao and other researchers. Yao and other applications used PD-1 knockout and spinal cord injury model, reveal that PD-1 knockout promoted direction of microglial cells and macrophage M1 polarization, and aggravated in ammatory response and neuronal damage (29).
Contrary to these observations, Bodhankar used stroke models and found that PD-L1 de ciency improved infarction volume, reduced in amed cells and in ammatory responses, and improved nerve function (36). It was not clear what causes this difference of study, and it might be related to differences in application of distinct animal models. Other scholars also reported that PD-1 signaling showed a protective role in persistent viral encephalitis. PD-1 signaling activation limited severity of in ammation during acute infection while it maintained a moderate in ammatory response during persistent infection and conduced to resistance to viral re-infection (37,38). It remained a question that if the PD-1 signaling pathway exhibited a "double-edged sword" effect in the different CNS diseases or different state of disease. In addition, it was noticed that PD-1 activation played a protective role by limiting in ammatory response by glial inhibition of T-cell function (39), which was worthy of in-depth study. Further clari cation of mechanism of PD-1/PD-L1 signaling pathway in glial cell activation and neuroin ammation shall be helpful to identify new drug intervention targets or establish new Parkinson's disease interventional treatment strategies.

Conclusions
By using PD-1 knockout mice and MPTP model, this study showed that PD-1 knockout state aggravated animal motor dysfunction of MPTP model by promoting microglial cell activation and neuroin ammatory reaction in the substantia nigra. Data of this study suggested that PD-1 signaling abnormality might be involved in PD pathogenesis or progression.